Our long-range goal is to understand the molecular mechanism of DNA replication and the processes which regulate DNA synthesis in the eukaryotic cell. This knowledge is fundamental to our understanding of such important questions as the mechanism of viral replication and the growth and differentiation of both normal and malignant cells. We will study the enzymes which function in DNA replication in the yeast Saccaromyces cerevisiae. In preliminary studies, we have identified a new yeast DNA polymerase. This enzyme will be extensively purified and its physical and enzymatic properties examined. We will produce monoclonal antibodies against the DNA polymerase. We anticipate that it will be difficult to obtain homogenous preparations of DNA polymerase. These antibodies will enable us to determine which protein bands are associates with DNA polymerase activity. We will search for factors (e.g. a primase) which function in the initiation of DNA chains, using single-stranded circular DNAs as templates. Since the initiation mechanism may require a specific yeast DNA sequence, we will test both in vitro and in vivo the ability of M13 yeast recombinant DNAs to serve as templates in the SS to RF reaction. We will attempt to develop a crude cell-free system for the replication of double-stranded DNA. By using a recombinant DNA plasmid which contains an origin for yeast DNA replication, a splasmid which can replicate in the yeast cell, we will determine whether replication in vitro requires a specific origin sequence. The requirements for various DNA synthesis gene products will be tested. The cell-free system may provide an in vitro complementation assay for these gene products. Ultimately, we hope to purify and characterize each of the proteins required for replication in this relatively simple model system.